downregulated apoptosis and autophagy after …

Paquet, C., Nicoll, J. A., Love, S., Mouton-Liger, F., Holmes, C.,Hugon, J., & Boche, D. (2017). DOWNREGULATED APOPTOSISAND AUTOPHAGY AFTER ANTI-Aβ IMMUNOTHERAPY INALZHEIMER'S DISEASE. Brain Pathology.https://doi.org/10.1111/bpa.12567

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https://doi.org/10.1111/bpa.12567

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DOWNREGULATED APOPTOSIS AND AUTOPHAGY AFTER

ANTI-Aβ IMMUNOTHERAPY IN ALZHEIMER’S DISEASE

Claire Paquet1,2,3 ; James AR Nicoll

4,5; Seth Love

6; François Mouton-Liger

2,7;

Clive Holmes4,8

; Jacques Hugon1,2,3

; Delphine Boche4

1 INSERM, U942, F-75010, Paris, France

2 University of Paris Diderot, Sorbonne Paris Cité, UMRS Inserm 942, F-75010, Paris, France

3 Centre de Neurologie Cognitive/Centre Memoire de Ressources et de Recherches Paris Nord Ile de France AP-

HP, Hôpital Lariboisière, F-75010, Paris, France

4 Clinical Neurosciences, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton,

Southampton, United Kingdom

5 Department of Cellular Pathology, University Hospital Southampton NHS Foundation Trust, Southampton,

United Kingdom

6 Department of Neuropathology, Institute of Clinical Neurosciences, School of Clinical Sciences, University of

Bristol, Bristol, United Kingdom

7. Inserm, U1127, Institut du Cerveau et de la Moelle épinière, ICM, F-75013, Paris, France

8 Memory Assessments and Research Centre, Moorgreen Hospital, Southern Health Foundation Trust,

Southampton United Kingdom.

Corresponding author:

Claire PAQUET, Centre de Neurologie Cognitive/Centre Mémoire de Ressources et de Recherches

Groupe Hospitalier Saint Louis-Lariboisière-Fernand Widal

200 rue du Faubourg Saint Denis 75475 PARIS Cedex, France

Phone: +33-1-40054313; Fax: +33-140054339;

E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/bpa.12567

This article is protected by copyright. All rights reserved.

mailto:[email protected]

http://orcid.org/0000-0002-2273-7316

http://orcid.org/0000-0002-5884-130X

Paquet et al. 2

Abstract

Aβ immunisation of Alzheimer’s disease (AD) patients in the AN1792 (Elan Pharmaceuticals) trial

caused Aβ removal and a decreased density of neurons in the cerebral cortex. As preservation of

neurons may be a critical determinant of outcome after Aβ immunisation, we have assessed the impact

of previous Aβ immunisation on the expression of a range of apoptotic proteins in post-mortem human

brain tissue. Cortex from 13 AD patients immunised with AN1792 (iAD) and from 27 non-immunised

AD (cAD) cases was immunolabelled for pro-apoptotic proteins implicated in AD pathophysiology:

phosphorylated c-Jun N-terminal kinase (pJNK), activated caspase3 (a-casp3), phosphorylated GSK3β

on tyrosine 216 (GSK3βtyr216), p53 and Cdk5/p35. Expression of these proteins was analysed in

relation to immunisation status and other clinical data. The antigen load of all of these pro-apoptotic

proteins was significantly lower in iAD than cAD (p < 0.0001). In cAD, significant correlations (p <

0.001) were observed between: Cdk5/p35 and GSK3βtyr216; a-casp3 and Aβ42; p53 and age at death. In

iAD, significant correlations were found between GSK3βtyr216 and a-casp3; both spongiosis and

neuritic curvature ratio and Aβ42; and Cdk5/p35 and Aβ-antibody level. Although neuronal loss was

increased by immunisation with AN1792, our present findings suggest downregulation of apoptosis in

residual neurons and other cells.

Keywords: Alzheimer, treatment, anti-amyloid immunotherapy, brain, neurons, impact.


Paquet et al. 3

INTRODUCTION

Alzheimer’s disease (AD) is characterized by the accumulation of β-amyloid (Aβ) peptide and

hyperphosphorylated tau protein, and eventually synaptic and neuronal loss. The pathophysiology of

the neuronal death remains unclear and controversial. Neuropathological studies have provided

evidence of apoptotic neuronal death compatible with the slow progression of neuronal degeneration

(15, 27, 32), in addition to possible deregulated autophagic activity (3, 14, 16, 24, 44). Apoptosis is a

sequence of programmed events leading to the activation of caspases and cell disintegration (15, 27,

32), whereas autophagy is an intracellular catabolic process leading to the removal of aggregated

proteins within cells (22, 28, 38). Both autophagy and apoptosis are highly regulated, play critical

roles in tissue homeostasis, and tend to be upregulated in response to extracellular or intracellular

stress and in neurodegenerative diseases (26). In AD, both processes have been extensively studied but

their contribution to neuronal death remains unclear. Apoptotic cell death in AD may result from an

imbalance between pro- and anti-apoptotic proteins (15). The expression of several pro-apoptotic

kinases such as activated GSK3β phosphorylated at tyrosine 216 (GSK3βtyr216) (1, 6, 37), pPKR (6, 7,

10, 29, 33, 34, 36), pJNK (9, 18, 42, 43), p53 (8) and activated caspase-3 (a-casp3) (2, 15, 17, 41) is

increased in AD brains. In AD, autophagic activity is increased but may be dysfunctional, with failure

of substrate clearance reflected by the presence of vacuoles (3, 14, 16, 24, 44).

Active Aβ42 immunisation (AN1792, Elan Pharmaceuticals) in AD patients led to Aβ removal (19, 30,

31) associated with a decrease in phosphorylated tau (pTau) (4), long-term down-regulation of

inflammation (46), reduction in the number of neurons and reduced neuritic abnormalities (34, 39). To

investigate possible mechanisms underlying the observed neuronal loss after immunotherapy, we have

explored the expression of apoptotic and autophagic proteins in the unique cohort of immunised AD

patients from the AN1792 trial.


Paquet et al. 4

MATERIALS AND METHODS

Case selection

Immunised AD cases (iAD)

The brains of clinical AD patients enrolled in the initial Elan Pharmaceuticals Aβ immunisation trial

AN1792 (19) were obtained following consent to post-mortem neuropathology. The study received

ethical approval from Southampton and South West Hampshire Local Research Ethics Committees

(Reference No: LRC 075/03/w). Thirteen post-mortem brains in which the cause of the dementia was

confirmed as AD neuropathologically were included in this study. All patients had received Aβ42 plus

adjuvant and had died between 4 and 162 months after the first immunisation (mean 72.8 months,

median 63 months), with Braak tangle stage V/VI disease, as previously described (34) (Table 1). The

post-mortem delay was between 6 and 48 hours (mean 18.5 hours; median 6 hours). In addition to

dementia, the most common clinical diagnoses recorded in the death certificate were

bronchopneumonia, cerebrovascular accident and myocardial infarction. Other diagnoses included

ruptured aortic aneurysm, pulmonary embolism, carcinoma of the breast, carcinoma of the bronchus,

and carcinoma of the pancreas. Neurodegenerative pathology was assessed by standard histological

methods including haematoxylin and eosin (H&E), Luxol fast blue/cresyl violet and modified

Bielschowsky silver impregnation. Selected sections were immunolabelled for Aβ, tau, α-synuclein

and TDP43 to confirm AD.

Non-Immunised AD cases (cAD)

Twenty-seven AD cases provided by the South West Dementia Brain Bank (SWDBB, Bristol, UK)

were identified and used as a control unimmunised AD cohort (supplementary Table 1). All cAD cases

had a clinical diagnosis of AD made during life by an experienced clinician, a Mini-Mental State

Examination score of <17 prior to death and satisfied post-mortem neuropathological Consensus

Criteria for Alzheimer’s disease (20). The post-mortem delay was between 9 and 110 hours (mean 39

hours, median 26 hours). The immunised and control AD cases were matched as closely as possible

for age, gender, duration of dementia and APOE genotype (Table 1). The SWDBB tissue was used


Paquet et al. 5

under the ethical approval from North Somerset and South Bristol Hampshire Local Research Ethics

Committees (Reference No: REC 08/H0106/28+5).

Immunohistochemistry

Middle temporal gyrus, usually markedly affected by AD pathology, was investigated in this study.

Four-µm sections of formalin-fixed paraffin-embedded tissue from iAD and cAD cases were

immunolabelled together in batches to ensure comparability of staining.

Primary antibodies and immunohistochemistry

To evaluate the impact of active AN1792 immunisation on apoptotic and autophagic pathways, we

explored by immunohistochemistry the expression of the following pro-apoptotic proteins: GSK3βtyr216

(polyclonal rabbit anti-phosphorylated GSK3βtyr216, #ab75745, Abcam) (6, 37), neuron-specific

activator of cyclin-dependent kinase 5 with its activator p35 (C-19 polyclonal rabbit anti-Cdk5/p35,

#sc-820, Santa Cruz) (12, 42), phosphorylated c-Jun N-terminal kinase (monoclonal rabbit anti-pJNK

Thr183/Tyr185, clone 81E11, #4668, Cell Signaling) (18, 45), p53 (monoclonal mouse anti-p53, clone

DO-1, #sc-126, Santa Cruz) (8), and a-casp3 (polyclonal rabbit anti-activated caspase 3 (Asp175), #

9661, Cell Signaling) (15, 40, 41); and of the autophagic proteins ATG5 (initial step) (polyclonal

rabbit anti-ATG5, #AP1812b, Abgent) and microtubule-associated protein light chain LC3-II (a

marker of the final stage reflecting efficient autophagic activity) (polyclonal rabbit anti- LC3-II,

#AP1801a, Abgent) (21, 22, 28). The specificity of the antibodies pJNK (18), GSK3βtyr216 (1), and

CDK5/p35 (21) was previously demonstrated. In order to demonstrate the specificity of the antibodies

p53, ATG5 and LC3II, we performed western blot on human brain tissue homogenates.

Immunohistochemistry was carried out by a standard method as previously described (1, 4, 5, 19, 30,

34, 46). Biotinylated secondary antibodies, normal serum and avidin-biotin complex were from Vector

Laboratories (Peterborough, UK). Immunodetection was performed using the avidin-biotin-peroxidase

complex method (Vectastain Elite ABC, UK) with 3,3’-diaminobenzidine (DAB) as chromogen and

0.05% hydrogen peroxide as substrate. All the sections were dehydrated before mounting in DePeX

(BDH Laboratory Supplies, UK). Sections from which the primary antibody was omitted were

included in each immunohistochemistry run.


Paquet et al. 6

Quantification of immunolabelling

Quantification was performed blind to the identity of the cases. Thirty fields of cortical grey matter at

objective magnification x20 were acquired for each case from the same anatomical regions in a zigzag

sequence along the cortical ribbon to ensure that all cortical layers were represented. Slides were

marked by the same neuropathologist to ensure consistency in the location of acquisition of the

images. Protein 'load' defined as the percentage of the field immunopositive for the marker of interest

was determined using ImageJ (developed by W.S. Rasband National Institutes of Health, Bethesda

MD, USA, version 1.47g), as in our previous studies (1, 4, 5, 19, 34, 46).

Statistical analysis

The normality of distribution of each marker across the cohort was assessed by examination of

quantile-quantile plots (not shown). Levels of each marker were compared between cAD and iAD

cases in two-sample two-sided t-tests or non-parametric Mann-Whitney U-tests (depending on the

normality of the data). In both groups, correlations were analysed by Pearson's or Spearman's test,

depending on the normality of distribution of the markers. We analysed the correlation between the

apoptosis and autophagy-associated markers and (i) indicators of disease severity and neuronal

integrity as reported in our previous published studies as follows: Aβ42 load, pTau load, tangles density

by image, dystrophic neurites, spongiosis, number neuronal NeuN+ density by image, neuritic

curvature ratio assessed by neurofilament immunohistochemistry, phosphorylated (p)PKR (a marker

of early neurodegeneration) (4, 19, 34, 46); and (ii) available clinical indicators of disease course and

antibody response – duration of dementia, survival time after immunisation, age at death, mean and

peak antibody level. The threshold for statistical significance was set at 5% for intergroup

comparisons and 1% for correlations, as determined by use of SPSS 21.0.


Paquet et al. 7

RESULTS

The immunolabelling of all of the antigens was neuronal, with additional labelling of glial cells for

some proteins as described in Table 2. Of note, the immunolabelling of activated-caspase 3 was

cytoplasmic with the nuclei of the stained neurons morphologically normal, without the karyorrhexis

classically associated with apoptosis.

The expression of all apoptotic kinases was significantly lower in iAD than cAD cases: a-casp3 load,

P<0.001; Cdk5/p35 load, P=0.013; p53 load, P<0.001; GSK3βtyr216 load, P<0.01; and pJNK, P<0.001

(Figure 1). Of the two autophagic markers examined, LC3-II load was significantly lower in iAD than

cAD (P<0.001) while ATG5 load did not differ between the cohorts (P=0.130, Figure 1).

The expression of apoptotic and autophagic markers was analysed for correlation with other aspects of

AD pathology (Aβ42 load, pTau load, dystrophic neurite counts, spongiosis, NeuN+ neurons and

curvature ratio) in the same anatomical region, and also with a range of clinical parameters (age,

gender, age at death, dementia duration, peak antibody, survival time). We did not observe any

modification in the distribution of the proteins between both cohorts except for the GSK3βtyr216, which

was detected mainly in granulo-vacular degeneration (GVD) in the iAD group but not in the cAD

group. To take account of possible variations in neuronal density, we also assessed the percentage of

all neurons that was immunopositive for a-casp3. This confirmed the striking decrease in neuronal

expression of a-casp3 in iAD compared with cAD (p<0.0001, data not shown).

In the cAD group, a-casp3 load correlated positively with Aβ42 (r==0.561, P=0.005), and Cdk5/p35

correlated positively with pGSK3βtyr216 (r==0.642, P<0.001) (Table 3). Comparison of present findings

with the clinical data revealed positive correlations between p53 and age at death (r==0.564, P=0.003),

and between LC3-II and dementia duration (r=0.691, P=0.001) (Table 3).

Within the iAD cohort, a-casp3 and GSK3βtyr216 correlated positively with severity of spongiosis, a

marker of neuropil degeneration (r=0.789, P=0.004 and r=0.761, P=0.007 respectively) (Table 2).

ATG5 correlated negatively with Aβ42 load (r==-0.845, P=0.001) and positively with the curvature

ratio (abnormal tortuosity of neuritic processes) (r==0.841, P=0.001) (Table 4). Cdk5/p35 correlated


Paquet et al. 8

positively with peak antibody titre (r=0.840, P<0.001) as well as with mean antibody titre (data not

shown) (Table 4).

No other correlation was observed in either group.

DISCUSSION

Our results suggest that active Aβ immunisation of AD patients modulates apoptosis and some

autophagic cellular signals, causing downregulation of apoptotic proteins and reduction in the final

stage of autophagy activity. The decrease of apoptotic protein expression after immunisation could

have several explanations: 1) Downregulation of apoptosis was a consequence of removal of Aβ,

consistent with several studies implicating Aβ-induced apoptosis in neuronal death in AD (6, 8). 2)

The reduction in apoptotic proteins may simply reflect the accelerated loss of damaged neurons after

immunotherapy, as previously reported by us (34), potentially leaving 'healthier' neurons less affected

by AD pathophysiology. However, the small magnitude of neuronal loss after immunotherapy (about

10%) could not be the sole explanation for the substantial decrease in apoptotic protein load (between

65% and 85%), and analysis of the percentage of all neurons that was immunopositive for a-casp3

confirmed the marked reduction in neuronal expression of this antigen in iAD. 3) Immunotherapy may

itself down-regulate apoptotic proteins. Further studies are needed to clarify the cellular and molecular

processes that underlie these findings.

The effects of autophagic proteins are less clear-cut. The reduction in LC3II suggests downregulation

of the later steps of autophagy, potentially explained by reduced metabolic requirement for autophagy

or perhaps an aborted or dysfunctional autophagic process. Restrictions on tissue availability did not

allow us to explore this mechanistically. Analysis in animal models may help to clarify the influence

of immunotherapy on autophagy.

The correlation between a-casp3 and Aβ42 in the cAD group, is in accordance with previous reports

implicating Aβ42 in neuronal apoptosis (6, 15). The link between Cdk5/p35 with GSK3βtyr216 is also


Paquet et al. 9

consistent with previous studies implicating these proteins in the pathophysiology of AD, particularly

in the phosphorylation of Tau protein (13, 23).

Strikingly different associations were observed in the immunised cohort. The relationship between a-

casp3, GSK3βtyr216 loads and the severity of spongiosis, a marker of neuropil degeneration, strengthen

the association between these pro-apoptotic proteins and the neuronal loss detected after immunisation

(34). This may explain the absence of clinical amelioration in these patients (19). Due to the nature of

the post-mortem study, investigating late-stage of the disease and treatment, we cannot exclude the

possibility that immunotherapy may have induced an early acute apoptotic phase followed by a more

quiescent phase several years after the treatment.

The relationship between p53 expression and age at death in the control Alzheimer’s cohort is

consistent with the documented association between apoptosis and increasing age (11). The increase in

LC3-II with dementia duration may be part of a pro-survival adaptive response by neurons and glia to

minimise neurodegeneration (14). After immunisation, the anti-Aβ immune response (mean and peak

Aβ antibody titre) was strongly associated with Cdk5/p35 expression. Cdk5/p35 signalling is known to

promote microglial phagocytosis of fibrillar Aβ (25), and the present data are in keeping with the

enhanced Aβ clearance by phagocytic microglia in the immunised patients who developed an immune

response (19, 35, 46). However, it should be noted that the highest Cdk5/p35 level in the immunised

cohort was much lower than that in the control group, consistent with the down-regulation of

microglial activation that occurs when Aβ has been completely removed (46).

This study has some limitations, inherent in the use of post-mortem tissue. As previously reported (1,

4, 5, 19, 30, 34, 46), the number of placebo immunisation cases from which brains could be obtained

(n=1) was far too low to provide useful data for statistical analysis and thus our study used AD brains

from patients who were not included in a protocol of immunotherapy, although they were matched as

closely as possible to the immunised cohort. Furthermore, this was a retrospective observational study

rather than a prospective experimental study, which limited the range of methodological approaches

and the comparability of clinical findings. Because this was an end-stage study, it was not possible to


Paquet et al. 10

explore the temporal relationship between markers of apoptosis or autophagy and neuronal loss, and

analysis was limited to assessment of the late-stage consequences of immunisation.

In summary, in this unique human brain series from the first anti-Aβ42 trial, our results suggest that

anti-Aβ42 immunisation downregulates the expression of several pro-apoptotic proteins in the brain.

Whilst these changes might be expected to be beneficial, the absence of cognitive benefit suggests that

they occur too late in the disease process or that other mechanisms are responsible for the neuronal

death.


Paquet et al. 11

Abbreviations

a-casp3 activated caspase 3

AD Alzheimer’s disease

ATG5 autophagy-related gene 5

Aβ β-amyloid

CDK5 cyclin dependent Kinase 5

GSK3βtyr216 glycogen Synthetase Kinase 3 phosphorylated at tyrosine 216

iAD immunised Alzheimer’s Disease brains

JNK c-Jun N Terminal Kinase

LC3 microtubule-associated protein light chain 3

p53 tumor protein 53

PKR double-stranded RNA dependent protein kinase

iAD immunised Alzheimer’s disease brains

cAD non-immunised Alzheimer’s disease brains

pTau phosphorylated tau

Ethical approval and consent to participate

The study received ethical approval from the Southampton and South West Hampshire Local Research

Ethics Committees, Reference No. LRC 075/03/w for the use of the iAD cohort. The cAD cases were

provided under the SWDBB Ethics (Research Ethics Committee Reference No. 08/H0106/28+5).

Competing interest

Prof. PAQUET is member of the International Advisory Boards of Lilly and is involved as

investigator in several clinical trials for Roche, Esai, Lilly, Biogen, Astra-Zeneca, Lundbeck

Prof. NICOLL is or has been a consultant/advisor relating to Alzheimer immunisation programmes for

Elan Pharmaceuticals, GlaxoSmithKline, Novartis, Roche, Janssen, Pfizer, Biogen.

Prof. HUGON is investigator in several passive anti-amyloid immunotherapies and other clinical trials

for Roche, Eisai, Lilly, Biogen, Astra-Zeneca, Lundbeck.


Paquet et al. 12

Prof LOVE, Prof HOLMES, Dr BOCHE and Dr MOUTON-LIGER declare that they have no conflict

of interest.

Funding

This study was supported jointly by the Fondation Philippe Chatrier (Paris, France), Alzheimer

Research UK (ART/PG2006/4 and ART-EXT2010-1) and Medical Research Council UK

(G0501033).

Author’s contributions

Claire PAQUET designed the study, performed the immunohistochemistry experiments, collected and

analysed the data and prepared the manuscript.

Delphine Boche analysed and interpreted the data and prepared the manuscript.

Seth Love provided the cAD cases from SWDBB and was involved in the preparation of the

manuscript.

Clive Holmes provided the clinical data.

François Mouton-Liger performed Western blot to control for the specificity of the antibodies and

prepared the manuscript.

Jacques Hugon advised on the relationship between different apoptotic kinases in Alzheimer’s’

disease.

James Nicoll provided immunised AD brains and was involved in the preparation of the manuscript.

All co-authors provided input and critically revised the paper.

"All authors read and approved the manuscript."

ACKNOWLEDGMENTS

All authors had full access to all data and CP and DB have final responsibility for the decision to

submit the report for publication.


Paquet et al. 13

We thank the patients who were involved in this study and their careers. We thank all donors, the

president and scientific committee of Fondation Philippe Chatrier. Vivienne Hopkins, David

Wilkinson, Anthony Bayer, Roy Jones and Roger Bullock enrolled patients in the original trial. Jim

Neal provided 2 immunised cases from Cardiff. We would like to thank the South West Brain

Dementia Brain Bank (SWDBB) for providing tissue for this study. The SWDBB is supported by

BRACE (Bristol Research into Alzheimer’s and Care of the Elderly), Brains for Dementia Research

and the Medical Research Council. The Neuropathology Section, Department of Cellular Pathology,

University Hospital Southampton NHS Foundation Trust, the Histochemistry Research Unit, and the

Biomedical Imaging Unit of the Faculty of Medicine, University of Southampton facilitated tissue

processing, staining and analysis. Staff at Elan Pharmaceuticals made available original clinical trial

data.


Paquet et al. 14

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Table 1 Characteristics of the immunised (iAD) and non-immunised (cAD) Alzheimer’s disease cohorts

ID case Gender Age Braak stage

Dementia

duration

(years)

APOE status

Mean antibody

response (ELISA

units)

Survival time from

1st injection

(months)

Post-mortem

delay (hours)

iAD1 F 74 VI 6 3.4 1:119 20 48

iAD2 M 83 V 11 3.3 <1:100 4 6

iAD3 M 63 VI 6 3.3 <1:100 41 6

iAD4 F 71 VI 10 3.3 1:4072 44 24

iAD5 M 81 VI 7 3.4 1:1707 57 6

iAD6 M 82 VI 6 3.4 1:4374 60 24

iAD7 M 63 VI 10 3.4 1:6470 64 6

iAD8 M 81 VI 11 4.4 1:491 63 ?

iAD9 F 88 VI 11 3.3 1:137 86 24

iAD10 M 88 VI 12 3.4 1:142 94 6

iAD11 F 89 VI 15 3.4 1:142 111 ?

iAD12 F 86 VI 13 4.4 <1:100 141 6

iAD13 F 75 VI 19 ? 1:221 162 48

cAD

(n=28)

15F:13M 63-88 V/VI 3-17 21ε4+:6 ε4- n/a n/a mean 39

median 26

n/a: non-applicable

?: unknown


Table 2: Topographical distribution of the apoptotic and autophagic proteins.

cAD Neurons Glial cells

Cytoplasm Nuclear Cytoplasm Nuclear

a-casp3 + - + -

Cdk5/p35 + - + -

pJNK + - + -

GSK3βtyr216 + + - -

P53 + - - -

LC3 + - + -

ATG5 + - - +

iAD Neurons Glial cells

Cytoplasm Nuclear Cytoplasm Nuclear

a-casp3 + - - -

Cdk5/p35 + - + -

pJNK + - - -

GSK3βtyr216 + + - -

P53 + - - -

LC3 + - - -

ATG5 + - - +


Table 3: Results of correlation analyses within the non-immunized AD control group

pJNK Cdk5/p35 p53 a-casp3 GSK3βtyr216 ATG5 LC3-II

Aβ42 r=0.141

p=0.483

r=-0.238

p=0.232

r=0.142

p=0.497 r=0.561**

p=0.005

r=-0.079

p=0.696

r=-0.173

p=0.399

r=-0.346

p=0.090

ptau r=-0.228

p=0.252

r=0.178

p=0.374

r=0.052

p=0.804

r=-0.224

p=0.303

r=0.365

p=0.061

r=-0.214

p=0.295

r=0.060

p=0.777

tangles r=-0.088

p=0.662

r=0.092

p=0.648

r=-0.254

p=0.221

r=-0.070

p=0.750

r=0.008

p=0.970

r=-0.387

p=0.050

r=-0.046

p=0.828

dystrophic neurites r=0.157

p=0.433

r=0.001

p=0.998

r=0.094

p=0.655

r=0.068

p=0.758

r=-0.010

p=0.959

r=-0.235

p=0.248

r=0.027

p=0.898

spongiosis r=-0.181

p=0.365

r=0.404

p=0.037

r=0.048

p=0.818

r=-0.327

p=0.128

r=0.166

p=0.409

r=0.231

p=0.256

r=0.084

p=0.690

NeuN r=0.008

p=0.971

r=-0.039

p=0.860

r=0.413

p=0.063

r=-0.118

p=0.610

r=0.361

p=0.090

r=0.232

p=0.298

r=0.160

p=0.489

NFP curvature ratio r=-0.042

p=0.837

r=0.180

p=0.369

r=0.182

p=0.383

r=-0.059

p=0.790

r=0.174

p=0.384

r=-0.055

p=0.788

r=0.134

p=0.524

pPKR r=-0.267

p=0.178

r=0.085

p=0.673

r=-0.081

p=0.701

r=0.094

p=0.670

r=0.337

p=0.085

r=0.110

p=0.593

r=-0.075

p=0.723

pJNK r=0.426

p=0.027

r=0.055

p=0.792

r=0.177

p=0.419

r=0.311

p=0.115

r=-0.226

p=0.266

r=0.202

p=0.334

Cdk5/p35 r=0.277

p=0.18

r=-0.146

p=0.505

r=0.648**

p<0.001

r=-0.196

p=0.338

r=0.300

p=0.144

p53 r=0.172

p=0.457

r=0.280

p=0.175

r=-0.055

p=0.795

r=0.319

p=0.120

a-casp3 r=-0.136

p=0.536

r=-0.492

p=0.020

r=-0.157

p=0.496

GSK3βtyr216 r=-0.01

p=0.927

r=0.128

p=0.542

ATG5 r=-0.062

p=0.770

Age at death r=0.210

p=0.294

r=0.289

p=0.144 r=0.564**

p=0.003

r=0.389

p=0.0670

r=0.438

p=0.022

r=-0.287

p=0.156

r=0.220

p=0.291

Dementia duration r=0.057

p=0.796

r=0.372

p=0.080

r=0.388

p=0.082

r=-0.062

p=0.795

r=-0.008

p=0.970

r=0.049

p=0.830

r=0.691

p=0.001

Peak antibody

r=0.033

p=0.914 r=0.840**

p<0.001

r=-0.175

p=0.569

r=-0.431

p=0.142

r=-0.284

p=0.348

r=0.459

p=0.115

r=-0.386

p=0.193

Survival time r=0.455

p=0.119

r=0.162

p=0.590

r=-0.077

p=0.802

r=0.252

p=0.406

r=0.446

p=0.126

r=0.280

p=0.354

r=0.568

p=0.043

Bold: ** correlation significant at the 0.01 level (2-tailed).


Table 4: Results of correlation analyses within the immunized AD control group

pJNK Cdk5/p35 p53 a-casp3 GSK3βtyr216 ATG5 LC3-II

Aβ42 r=-0.237 p=0.482

r=-0.491 p=0.125

r=-0.361 p=0.276

r=0.413 p=0.207

r=0.324 p=0.331

r=-0.845**

p=0.001

r=0.484 p=0.131

ptau r=0.397

p=0.226

r=0.082

p=0.811

r=0.164

p=0.629

r=0.089

p=0.794

r=-0.231

p=0.494

r=0.036

p=0.915

r=-0.174

p=0.610

tangles r=0.301 p=0.368

r=0.464 p=0.151

r=-0.050 p=0.883

r=-0.089 p=0.796

r=-0.207 p=0.541

r=0.155 p=0.650

r=-0.507 p=0.112

dystrophic neurites r=0.037

p=0.915

r=-0.246

p=0.466

r=-0.165

p=0.628

r=0.667

p=0.025

r=0.654

p=0.029

r=-0.269

p=0.424

r=0.547

p=0.082

spongiosis r=0.479

p=0.136

r=0.009

p=0.979

r=-0.087

p=0.800 r=0.789**

p=0.004

r=0.761**

p=0.007

r=-0.055

p=0.873

r=0.128

p=0.708

NeuN r=0.662

p=0.037

r=-0.353

p=0.318

r=0.107

p=0.769

r=0.691

p=0.027

r=0.337

p=0.340

r=0.170

p=0.638

r=0.055

p=0.880

NFP curvature ratio r=0.448

p=0.167

r=0.377

p=0.253

r=0.194

p=0.568

r=-0.152

p=0.656

r=0.137

p=0.687 r=0.841**

p=0.001

r=-0.418

p=0.201

pPKR r=0.201

p=0.577

r=-0.564

p=0.090

r=0.213

p=0.555

r=0.297

p=0.405

r=0.258

p=0.471

r=-0.176

p=0.627

r=0.701

p=0.024

pJNK r=0.11

p=0.720

r=0.083

p=0.788

r=0.534

p=0.060

r=0.078

p=0.801

r=0.529

0 p=.063

r=-0.300

p=0.319

Cdk5/p35 r=-0.223

p=0.464

r=-0.049

p=0.873

r=0.102

p=0.739

r=0.363

p=0.223

r=-0.342

p=0.253

p53 r=0.052 p=0.865

r=-0.233 p=0.444

r=0.165 p=0.589

r=0.268 p=0.375

a-casp3 r=0.546 p=0.054

r=-0.165 p=0.590

r=-0.069 p=0.823

GSK3βtyr216 r=-0.108

p=0.726

r=-0.218

p=0.474

ATG5 r=-0.303 p=0.314

Age at death r=0.512

p=0.074

r=-0.502

p=0.08

r=-0.029

p=0.925

r=-0.080

p=0.795

r=0.082

p=0.791

r=0.337

p=0.261

r=-0.262

p=0.388

Dementia duration r=0.119

p=0.700

r=-0.125

p=0.684

r=-0.297

p=0.324

r=0.134

p=0.661

r=0.178

p=0.560

r=0.008

p=0.978

r=-0.292

p=0.333

Peak antibody r=0.033 p=0.914

r=0.840**

p<0.001

r=-0.175 p=0.569

r=-0.431 p=0.142

r=-0.284 p=0.348

r=0.459 p=0.115

r=-0.386 p=0.193

Survival time r=0.455 p=0.119

r=0.162 p=0.590

r=-0.077 p=0.802

r=0.252 p=0.406

r=0.446 p=0.126

r=0.280 p=0.354

r=0.568 p=0.043

Bold: ** correlation significant at the 0.01 level (2-tailed).


Figure 1: On the left, illustration of the immunolabeling of pro-apoptotic and autophagic proteins as observed in Alzheimer's disease. On the right, quantification of the proteins in the non-immunised AD (cAD) compared to immunised AD (iAD) cases showing a significant decrease in all apoptotic proteins and of LC3II

after immunisation. Scale bar = 50µm.

99x279mm (300 x 300 DPI)


downregulated apoptosis and autophagy after …

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